Project Summary Cryogenic electron microscopy (cryo-EM) is a rapidly emerging technology for structural biology. While excitement and demand for the technology are high, the pace of training cryo-EM practitioners lags far behind. There are two major hurdles to the development of effective training programs: (1) potential learners come from a wide range of backgrounds and career stages, from graduate students to established structural biologists; and (2) learning cryo-EM requires many hours of hands-on ?behind the wheel? training on specialized, expensive equipment that is often unavailable to learners at their home institutions. While some hands-on training schools exist for cryo-EM, they train only a handful of students a year, while demand for the technique requires orders of magnitude more. Workshops enroll more students, but are too short to teach all the necessary theory and lack a hands-on component. To train a skilled workforce of cryo-EM practitioners, here I propose the framework for a comprehensive online curriculum, based on my successful ?Getting Started in Cryo-EM? video lectures, covering the theory and practice of all major cryo-EM modalities. The curriculum will target two groups: (1) the growing number of non-practitioners who want to learn about cryo-EM but will not operate a microscope themselves and (2) future cryo-EM practitioners. For non-practitioners such as professors who want to add a discussion of cryo-EM to their courses, I will develop four ?Teacher Packs,? containing video lectures and associated instructor resources (flipped class discussion questions, review slides, homework problems, and exam questions), each targeted to a different course format. For practitioners, together with Matthijn Vos and Tamir Gonen I will develop modular ?Units? of theoretical and lab demonstration videos, accompanied by training manuals and certification tests. Units will cover general cryo-EM theory and operation, single-particle reconstruction, tomography, micro-electron diffraction / electron crystallography, and advanced topics and troubleshooting for facility managers, and can be combined to meet the specific needs of the learner. In conjunction with supervised training at a user's home facility or cryo-EM service center, the curriculum will offer everything a student needs to understand the theory of, and begin practicing, their desired cryo-EM technique.